Enterotoxigenic Escherichia coli (ETEC) are among the primary causes of infantile and traveler's diarrhea. Despite the number and severity of ETEC infections worldwide, at present no licensed vaccine is available for at-risk individuals. We have developed a live attenuated E. coli vaccine strain by deleting or modifying major virulence determinants of a WT O157:H7 Enterohemorrhagic E.coli (EHEC) isolate. We now propose to use this strain as a vector to express ETEC antigens. We hypothesize that by delivering critical ETEC virulence determinants (adhesins and toxin components) to the mucosal immune system in an attenuated, non-invasive live vaccine strain, effective systemic and local immune responses can be generated which will protect against ETEC challenge. We will use an established mouse intranasal model of immunization and pathogen challenge to determine safety and immunogenicity and protective efficacy of our vaccine strains. Our first aim is to prepare derivatives of our attenuated attaching/effacing enterohemorrhagic E. coli (EHEC) vaccine that efficiently express components of two critical ETEC antigens, the CFA/I colonization factor and the heat-labile enterotoxin (LT). Our second aim is to determine a safe immunizing dose of the attenuated vaccine strains by monitoring immunized mice for adverse effects. Our third aim is to measure serum and mucosal immune responses following intranasal immunization with the vaccine constructs in order to determine the correlates of protection, and also of reactogenicity. Our fourth aim is to determine the level of protective efficacy resulting from immunization with the vaccine constructs using an established intra-pulmonary challenge model which we have previously used to determine the efficacy of other ETEC vaccine constructs. To achieve these ends, the expression of ETEC antigens by the vaccine constructs will be demonstrated by SDS-PAGE, immunoblotting, and bacterial agglutination. We will assess the safety of the vaccine constructs by noting any adverse clinical signs, and gross and histological changes at necropsy and by performing complete blood cell counts and blood chemistry in the vaccinated animals. We will assess the level of both systemic and local immune responses in the vaccinated mice by ELISA, cytokine responses and functional assays. We will determine protective efficacy by intranasal challenge with a lethal dose of the wild-type ETEC strain, as well as by measuring ETEC bacterial clearance from the lungs of the vaccinated mice. Development of safe and effective vaccines directed against ETEC strains should have significant public health benefit for infants in developing countries and for international travelers. PUBLIC HEALTH RELEVANCE: Enterotoxigenic E. coli (ETEC) are important bacterial pathogens causing worldwide morbidity and mortality. ETEC infections are important causes of death in infants and children under the age of five years in developing countries. ETEC are also the leading cause of diarrhea in travelers to high-risk areas of the world. Despite the fact that virulence factors of ETEC are well understood, and although in the last few decades there have been several potential ETEC vaccines tested in volunteer trials and field studies, no safe and effective vaccine is yet available for at-risk individuals. The development of safe and effective, live attenuated ETEC vaccines should have great public health significance for infants in developing countries and for international travelers.